Whole cell vaccines are often immunogenic without added adjuvants, but production costs and safety issues have prompted the development of protein-based alternatives. However, protein-based vaccine design is challenged by limited immunogenicity and the necessity of adjuvants, few of which are currently available for human use. We have developed a peptide-based platform that self-assembles, and when these peptides are fused to short peptide antigens, they elicit substantial antibody responses in mice to the short peptide antigens without the need for additional adjuvant (1). Antibody responses have been broad, including all IgG subclasses and IgM, and they have been strongly persistent for over 36 weeks in mice. These findings suggest these self-assembling materials as attractive novel candidates for vaccine development. We propose to expand on the capability of these materials to display full-length proteins, and to test them as a protective modality for clinically important community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Antibodies (Abs) against the S. aureus alpha-hemolysin (Hla), a pore-forming cytotoxin, have been shown to protect against lethal pneumonia and skin infection in mice (2-6). In the first specific aim, we will assess the ability of the Hla conjugated to the self-assembled peptide platform to elicit anti-Hla Ab production. To this end, we will utilize a novel protein conjugation technique based on a mutant version of the fungal cutinase enzyme to covalently bond cutinase-Hla fusion proteins to suicide phosphonate ligands on the self- assembling peptide platform (7). Immunoelectron microscopy and analytical ultracentrifugation will be used to validate fibrillar assembly and epitope availability, and we will assess the ability of the Hla assemblies to stimulate anti-Hla Ab production in mice. These studies are designed to provide proof-of-principle that these self-assembling peptides can display full-length proteins in a highly immunogenic manner. In the second specific aim, we will assess protection afforded by Hla self-assemblies against CA- MRSA infection. We will utilize two CA-MRSA infection models in mice, a pulmonary and a skin infection model. Both models have been developed and utilized extensively by our collaborative team. Measures of protection include survival, S. aureus CFU recovered from the lung and skin, histopathology, and size of dermonecrotic skin lesions. We will further determine the extent to which Hla-specific antibodies elicited by the Hla-peptide vaccination are the basis for protection, by transferring sera from immunized mice into naive mice infected with CA-MRSA. These studies are designed to provide proof-of-principle that these self-assembling peptides presenting Hla can elicit antibody responses that are protective against CA-MRSA. Success in these studies will provide us with insights into the utility of these peptide assemblies as highly engineered vaccines for the prophylactic control of diseases caused by pathogens, and as a modular platform for designing sophisticated immune-modulating reagents. PUBLIC HEALTH RELEVANCE: Vaccination is the most effective means for the prevention of disease from multiple pathogens, but numerous challenges are faced by both whole cell vaccines and protein-based alternatives, which require adjuvants to enhance their efficacy. We have developed a self-assembling peptide system that, when coupled to peptide antigens, raises strong antibody responses to the peptide antigens in mice, without the need for additional adjuvant. In this proposal we seek to expand the capability of these materials to display clinically important full-length proteins and to conduct mechanistic studies to understand the basis of their immunogenicity, with the anticipation that success in these goals will provide us with critical insights into utility of these peptide systems for vaccination against pathogens.